We have demonstrated using I-125-ECGF autoradiography that heparin markedly inhibits proteolytic digestion of ECGF by trypsin and other proteases. This property was lost after thermal denaturation of ECGF, suggesting a heparin:ECGF structural interaction rather than a heparin:trypsin interaction is responsible for the trypsin resistance of ECGF. Heparin was better able to protect ECGF from subsequent trypsin digestion after thermal denaturation in the presence of heparin as compared with thermal denaturation in the absence of heparin suggesting that heparin ameliorates ECGF denaturation and provides conformational stability to the polypeptide growth factor. The stabilizing effect of heparin was dependent upon the concentration of heparin as well as temperature and duration. Autoradiography of I-125-ECGF incubated with human umbilical vein endothelial cells demonstrated near complete inhibition of proteolytic digestion of ECGF when the incubation was performed in the presence of heparin. These data suggest that the mechanism of the heparin-induced human endothelial cell phenotype involves the protection of ECGF by heparin against inactivation by endothelial cell-derived proteolytic enzymes.